This chapter describes Molecular Probes affinity reagents for protein detection. These reagents are primarily conjugated and unconjugated antibodies, augmented by avidin and streptavidin conjugates for detecting biotinylated proteins and lectin conjugates for detecting glycoproteins. Collectively, they represent the combination of our premium fluorophores with our bioconjugation expertise to produce an unsurpassed range of products for histochemical and cytochemical analysis.
Despite the advent of competing approaches such as recombinant peptide tagging and mass spectrometry, antibody-based detection remains the most broadly applicable means of localizing and quantitating specific proteins in complex samples. Although the primary emphasis is on proteins, some of these reagents, particularly avidin and streptavidin conjugates, have important applications for the detection of nucleic acids and other biomolecules. Non-antibody based approaches for protein detection are discussed in Protein Detection and Analysis—Chapter 9. Within this chapter, Molecular Probes affinity reagents for protein detection are subdivided as described below.
Secondary Immunoreagents—Section 7.2
Labeled secondary antibodies form stable and specific complexes with unlabeled primary antibodies (Figure 7.1.1), providing the foundation for most immunofluorescence microscopy protocols. Molecular Probes secondary antibody offering provides an extensive selection of labels that include our superior Alexa Fluor dyes, phycobiliproteins, Alexa Fluor dye–phycobiliprotein tandem fluorophores, Qdot nanocrystals, biotin and enzyme labels (horseradish peroxidase and alkaline phosphatase). We also offer many options in terms of immunoreactivity, an essential consideration in avoiding confounding crossreactivity when performing simultaneous secondary immunodetection of two or more targets. Our labeled secondary antibody portfolio includes antibodies against IgG and IgM from several mammalian species, including various isotypes of mouse IgG, as well as against avian (chicken) IgY.
Figure 7.1.1 Schematic diagram of primary and secondary detection reagents. A) In primary detection methods, the target-specific molecule includes one or more detectable moieties, shown here as radiant orbs. B) In secondary detection methods, the target-specific molecule contains binding sites or haptens that can be selectively recognized by secondary detection reagents. For example, these sites might be antigenic epitopes that bind antibodies. Alternatively, the target-specific molecule might be conjugated to either biotin or fluorescent dyes, thereby creating a molecule that can be detected with any of our avidin and streptavidin conjugates or our anti–fluorescent dye antibodies (Antibodies, Avidins and Lectins—Chapter 7). As shown here, the target-specific molecule may contain multiple sites for binding the secondary detection reagent, thereby providing a simple system for amplifying the signal.
Zenon Technology: Versatile Reagents for Immunolabeling—Section 7.3
Molecular Probes Zenon antibody labeling technology uses reagents that are technically secondary antibody conjugates in rapid immunolabeling protocols that would normally require preparation or purchase of directly labeled primary antibodies. Rapid processing enabled by elimination of the secondary reagent incubation step in conventional immunolabeling protocols is vital when identifying cells for mRNA extraction and downstream genomic analysis. Elimination of the secondary reagent incubation step also enables the use of multiple antibodies derived from the same species in the same immunolabeling protocol.
Anti-Dye and Anti-Hapten Antibodies—Section 7.4
Antibodies to fluorophores and other nonfluorescent labels provide unique opportunities for signal enhancement and for correlated fluorescence and electron microscopy studies. For example, transplanted cells labeled with fluorescein-based tracers such as CFSE (C1157, V12883; Membrane-Permeant Reactive Tracers—Section 14.2) can be immunohistochemically detected using our anti–fluorescein/Oregon Green antibodies in situations where the fluorescein fluorescence signal is undetectable due to cellular dissemination in vivo. Dye/anti-dye antibody affinity pairs can also be used as alternatives to biotin/avidin detection methods in applications such as in situ hybridization and enzyme-linked immunosorbent assays (ELISAs).
Antibodies against Expression Tags—Section 7.5
This section presents a selected set of primary antibodies for detection of expression tags, including Green Fluorescent Protein (GFP) and Red Fluorescent Protein (RFP). The full range of Invitrogen primary antibodies can be surveyed using our online primary antibody search utility at www.invitrogen.com/handbook/antibodies. Direct labeling of primary antibodies can be accomplished via amine-reactive chemistry implemented in our comprehensive selection of convenient and easy-to-use protein labeling kits (Kits for Labeling Proteins and Nucleic Acids—Section 1.2). Alternatively, Zenon immunolabeling technology (Zenon Technology: Versatile Reagents for Immunolabeling—Section 7.3) can fulfill the same purposes and is less demanding in terms of antibody quantity and purity requirements.
Avidin and Streptavidin Conjugates—Section 7.6
Molecular Probes fluorophore-conjugated avidins and streptavidins are extensively used in conjunction with biotinylated oligonucleotides or biotinylated antibodies for nucleic acid hybridization analysis, immunohistochemistry and multicolor flow cytometry. Qdot nanocrystal–streptavidin conjugates used for the same purposes are described in Qdot Nanocrystals—Section 6.6. Section 7.6 also features our enzyme-labeled avidins and streptavidins, NeutrAvidin-coated microspheres and affinity matrices for capture and isolation of biotinylated proteins and nucleic acids. Biotin conjugates and the reagents and methods used for their preparation are described in Biotin and Hapten Derivatives—Chapter 4.
Lectins and Other Carbohydrate-Binding Proteins—Section 7.7
Lectins and certain other carbohydrate-binding proteins that bind to specific glycans are versatile primary detection reagents for histochemical and cytochemical analysis of glycoproteins and glycolipids. Complementary detection methods for glycoproteins based on Molecular Probes Click-iT labeling technology are described in Click Chemistry—Section 3.1 and Detecting Protein Modifications—Section 9.4.